De novo design of diverse small molecule binders and sensors using Shape Complementary Pseudocycles

Abstract

A general method for designing proteins to bind and sense any small molecule of interest would be widely useful. Due to the small number of atoms to interact with, binding to small molecules with high affinity requires highly shape complementary pockets, and transducing binding events into signals is challenging. Here we describe an integrated deep learning and energy based approach for designing high shape complementarity binders to small molecules that are poised for downstream sensing applications. We employ deep learning generated psuedocycles with repeating structural units surrounding central pockets; depending on the geometry of the structural unit and repeat number, these pockets span wide ranges of sizes and shapes. For a small molecule target of interest, we extensively sample high shape complementarity pseudocycles to generate large numbers of customized potential binding pockets; the ligand binding poses and the interacting interfaces are then optimized for high affinity binding. We computationally design binders to four diverse molecules, including for the first time polar flexible molecules such as methotrexate and thyroxine, which are expressed at high levels and have nanomolar affinities straight out of the computer. Co-crystal structures are nearly identical to the design models. Taking advantage of the modular repeating structure of pseudocycles and central location of the binding pockets, we constructed low noise nanopore sensors and chemically induced dimerization systems by splitting the binders into domains which assemble into the original pseudocycle pocket upon target molecule addition.

Figure 1.. Pseudocycle-based SC optimizing design method and target SMs.

(a) Diverse conformers of the SM of interest are docked into deep learning generated pseudocycles containing a wide array of central pockets(11), and the interface sequence optimized for high affinity binding using Rosetta or LgandMPNN. Top ranked designs are tested experimentally, and the backbones of the best hits and the docked poses are extensively resampled. Following sequence design, top ranked second round designs are experimentally tested (see Fig S1). (b) Because pseudocycles are constructed from modular repeating units which surround the central binding pocket, the binders can be readily transformed into sensors through multiple strategies. (c-f) Examples of first round design models for each target ligand.

Figure 2.. X-ray crystallography demonstrates accuracy of design approach.

(a) The crystal structure of CHD_r1 (gray) is very similar to its computational design model (teal). (b-c) The designed sidechain interactions with the three CHD hydroxyl groups and the buttressing hydrogen bonding network are closely recapitulated in the crystal structure (design is colored, crystal structure is in gray). (d) The ligand and the key interacting residues were well resolved with clear electron density. The protein backbone is shown in cartoons, and CHD and the key interacting side chains in sticks. Pink, ligand carbon atoms; red, oxygen; blue, nitrogen; white, polar hydrogen. The residue numbers of the key residues are labeled. Also see Fig S7a–c.

Figure 3.. Experimental characterization of selected designed binders from the affinity improving round.

(a-d) Nanomolar affinity CHD binders CHD_d1 to d4 (full list in Fig S9), (e) nanomolar binder for AMA (see Fig S12); (f) micromolar methotrexate binder, (g-h) two nanomolar T44 binders (full list at Fig S14). For each panel, from left to right, the design model, zoom in on the sidechain-ligand interactions, FP (or SPR in the case of AMA) binding measurements, and SEC traces. Kd values and error bars are from two independent experiments. Interacting side chains and ligands are shown in sticks, with oxygen, nitrogen, iodine, and polar hydrogen colored in red, blue, purple, and white, respectively. Key interactions are indicated by gray dashed lines. The cartoon of and sticks from helixes, sheets, and loops are colored in teal, magenta, and dark blue, respectively.

Figure 4.. Conversion of pseudocycle binders into ligand gated channels and CID systems.

(a-f) Ligand sensing de novo nanopore construction. (a) The three structural repeat units of CHD_r1 were inserted into three different loops in a 12 stranded de novo nanopore using inpainting to join the chains such that the central axes of binder and nanopore are aligned. The conductance of the original nanopore is ~220 pS (Fig S17a), and is not influenced by CHD. The conductance of binder-fused nanopore in the absence (b) and presence of CHD (c-e): In the absence of CHD (c), the pore fluctuates between a state with high conductance very similar to the unmodified pore and a low conductance state (c); in the presence of CHD the duration of the low conductance states is greatly increased (c-e); the longer record (c) and a single closure event (d) are shown for clarity, and the histogram of the current with and without ligand is shown in (e). Different currents, 0, 10, 15, 20 pA are marked out for clarity using dashlines in black, magenta, teal, and green, respectively in b-e. The gated nanopores are robust through multiple cycles of opening and closure (c), upon reversal of the voltage, the original high conductance state is restored (Fig S17b). (f) The conductance fluctuations of the binder-fused pore in absence of ligand likely reflect transient association of the 3 subunits; ligand binding stabilizes the associated state leading to prolonged blocking of the pore. (g-k) CID system construction. CHD binder, CHD_r1, was buttressed by diffusion of an outer ring of helices to increase the stability of split protein fragments (g & Fig S18). The crystal structure of the buttressed binder with ligand (h & Fig S7d–f) is in close agreement with the design model (i). To create a CID system, we split the buttressed binders into halves and redesign the protein-protein interface to increase solubility of the fragments and disfavor association in the absence of ligand. Characterization of CHD induced association of the split fragments by size exclusion chromatography (k) and mass photometry (j). Dimerization of the two split domains (A and B) in presence (first trace from top), but not the absence, (second trace from top) of ligand. The individual monomers do not dimerize in the presence(third and forth trace from top) or absence (fifth and sixth traces from the top) of ligands. N terminal gfp tags were fused to the monomers to facilitate detection by mass photometry.